Kranzhöfer A, Baker A H, George S J, Newby A C
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom.
Arterioscler Thromb Vasc Biol. 1999 Feb;19(2):255-65. doi: 10.1161/01.atv.19.2.255.
Degradation of the extracellular basement membrane is implicated in atherosclerosis, restenosis after angioplasty, and intimal thickening of vein grafts. Upregulation of metalloproteinase (MMP)-2 and MMP-9 accompanies neointima formation in cholesterol-fed rabbits, in rat and pig models of angioplasty, and in organ cultures of human saphenous veins. MMPs are inhibited by binding to tissue inhibitors of MMPs (TIMPs). Relatively little is known about their regulation in relationship to neointima formation; thus, we investigated TIMP expression in the organ culture model. Qualitative reverse transcriptase-polymerase chain reaction of mRNA extracted from veins showed that TIMP-1, TIMP-2, and TIMP-3 are each expressed before and after culture. Zymography revealed that TIMP-1 was the most abundant TIMP secreted and that its secretion increased dramatically between 0 to 2 and 12 to 14 days of culture. An enzyme-linked immunosorbent assay showed that TIMP-1 secretion increased from 3.2+/-1.5 (mean+/-SE) to 32+/-6 ng/mg wet weight per day (n=5, P<0.01). Immunocytochemical testing localized the increased expression of TIMP-1 to neointimal smooth muscle cells. Although less abundant, TIMP-2 secretion also increased from 0.8+/-0. 3 to 4.7+/-0.2 ng/mg wet weight per day (n=5, P<0.001), and tissue levels increased from 33+/-7 to 150+/-70 ng/mg wet weight (P<0.05). TIMP-2 was also immunolocalized to neointimal smooth muscle cells and their surrounding matrix. TIMP-3 was not secreted but was detected variably and constitutively in tissue extracts (160+/-120 and 170+/-100 ng/mg wet weight [n=9] on days 2 and 14, respectively). TIMP-3 was found in the cells and extracellular matrix of the media and adventitia before culture and to a lesser extent in the neointima after 14 days of culture. Rates of total TIMP secretion on day 14 exceeded those of MMP-2 and MMP-9 (10.6+/-1.9 and 15.6+/-2.3 ng/mg wet weight per day, respectively). Consistent with this, in situ zymography showed that MMP gelatinase activity was highly localized to cell bodies in the media and neointima. Secretion of TIMP-1 and TIMP-2 is greatly increased during neointima formation in human saphenous veins. TIMP-1 is readily released, whereas TIMP-2 remains partially attached and TIMP-3 exclusively attached to the extracellular matrix. Regulation of TIMP expression is therefore an important determinant of net MMP activity during neointima formation, restricting it to the pericellular environment.
细胞外基底膜的降解与动脉粥样硬化、血管成形术后再狭窄以及静脉移植物内膜增厚有关。在喂食胆固醇的兔子、大鼠和猪的血管成形术模型以及人隐静脉的器官培养中，金属蛋白酶（MMP）-2和MMP-9的上调伴随着新生内膜的形成。MMP通过与MMP组织抑制剂（TIMP）结合而受到抑制。关于它们在与新生内膜形成相关的调节方面了解相对较少；因此，我们在器官培养模型中研究了TIMP的表达。从静脉中提取的mRNA的定性逆转录聚合酶链反应表明，TIMP-1、TIMP-2和TIMP-3在培养前后均有表达。酶谱分析显示，TIMP-1是分泌最多的TIMP，其分泌在培养的0至2天和12至14天之间显著增加。酶联免疫吸附测定表明，TIMP-1的分泌从3.2±1.5（平均值±标准误）增加到每天32±6 ng/mg湿重（n = 5，P<0.01）。免疫细胞化学检测将TIMP-1的表达增加定位到新生内膜平滑肌细胞。虽然含量较少，但TIMP-2的分泌也从每天0.8±0.3增加到4.7±0.2 ng/mg湿重（n = 5，P<0.001），组织水平从33±7增加到150±70 ng/mg湿重（P<0.05）。TIMP-2也免疫定位到新生内膜平滑肌细胞及其周围基质。TIMP-3不分泌，但在组织提取物中可检测到且组成性存在（培养第2天和第14天分别为160±120和170±100 ng/mg湿重[n = 9]）。培养前在中膜和外膜的细胞及细胞外基质中发现TIMP-3，培养14天后在新生内膜中含量较少。第14天TIMP的总分泌率超过MMP-2和MMP-9（分别为每天10.6±1.9和15.6±2.3 ng/mg湿重）。与此一致，原位酶谱分析表明MMP明胶酶活性高度定位于中膜和新生内膜的细胞体。在人隐静脉新生内膜形成过程中，TIMP-1和TIMP-2的分泌大幅增加。TIMP-1易于释放，而TIMP-2仍部分附着，TIMP-3仅附着于细胞外基质。因此，TIMP表达的调节是新生内膜形成过程中MMP净活性的重要决定因素，将其限制在细胞周围环境。
